Piercing and incubation method of in planta transformation producing stable transgenic plants by overexpressing DREB1A gene in tomato (Solanum lycopersicum Mill.)

Cold is a major constraint for tomato growth and productivity; as it is a cold sensitive crop. DREB1A plays a key role in generating cold tolerance in tomato by regulating the response of multiple genes under chilling stress. In this study, cold tolerant gene (DREB1A) driven by Lip9 promoter, was transformed in three tomato genotypes through Agrobacterium tumefaciens, employing tissue culture independent transformation strategy. Overnight imbibed seeds of tomato were surface sterilized, 3-day old shoot apical meristem of the developing seedling were pierced and incubated for twenty minutes with A. tumefaciens strain EHA-105 having OD600 nm of 1.0. The treated seeds were co-cultivated with Agrobacterium for 2 days. The regenerated shoots were subjected to 35 mg/l hygromycin as a selection for a period of 2-3 weeks. The presence of DREB1A and hpt genes in the hygromycin resistant (T-0) transgenic plants was evaluated by PCR analysis. The transgene activity was detected in T-1 plants by Reverse Transcriptase Polymerase Chain Reaction and Southern blotting that showed the stable integration of the transgene to the next generation. Physiological analysis of T-2 transgenic plants depicted that increased expression of DREB1A induced only during chilling stress. After various chilling stresses, stomatal conductance, transpiration rate and relative water contents of transgenic lines were significantly higher than those of NT plants. While leaf osmotic potential of transgenic plants was lower as compared to NT plants. The established procedure is novel and can produce stable transgenic tomato plants in efficient manner by saving potential resources in terms of cost and time.